Human carcinoembryonic antigen (CEA), which is extensively expressed in the majority of human cancers is a potential target for specific immunotherapy using recombinant vaccines. Following immunizations of recombinant vaccinia-CEA (rV-CEA), enhancement of CEA-specific cytotoxic T lymphoctyes (CTL) responses was observed in patients. A CEA-specific T-cell line was established from a patient with metastatic colorectal carcinoma and cultured long-term in vitro. The cell line (designated V8T) was shown to be CD8+, CD2+/CD54+, CD45+/CD49d+, and CD11a+/CD58+. V8T cells lyse CAP-1 pulsed C1R:A2 cells. V8T did not lyse non-HLA-A2,CEA positive allogeneic carcinoma cells. Allogeneic HLA-A2 positive and CEA negative carcinoma cell could not serve as target for V8T cells while an allogeneic HLA-A2 and CEA positive carcinoma cell line was lysed. The cytotoxicity of V8T cell line was shown to be MHC class I restricted. The phenotype and specificity of V8T are stable after 22 stimulations (11 months). These studies also demonstrated significant difference in the precursor frequency of anti-tumor CTL pre- vs post-immunization. In an effort of study other potential CTL CEA epitopes, the ability of an additional human CEA epitope (CAP-2) to induce CEA-specific CTL was investigated. CAP-2 peptide is a 9-mer HLA-A2 binding CEA peptide. Two cytotoxic T-cell lines were established from a metastatic colorectal carcinoma patient immunized with rV-CEA. These two T-cell lines were shown to be CD8+/CD4-, or CD8+/CD4+ and CD56-. These CTLs lyse CAP-2 pulsed C1R:A2 cells and CEA positive carcinoma cells in a HLA-A2 restricted manner. These CTLs respond to CAP-2 with an enhanced production of IFN-gamma, TNF-alpha, and GM-CSF. We also investigated the ability of prostate specific antigen (PSA) epitopes to activate human cytotoxic T lymphocytes in vitro. Two novel HLA-A2 binding PSA epitopes (10 mers, designated PSA-1 and PSA-3), a novel HLA-A3 binding PSA epitope (9-mer, designated PSA-9) and a longer PSA peptide (30 mer, designated PSA-OP), containing PSA-1, PSA-3 and PSA-9 peptide sequences have been identified. CTLs were established, and the specificity of these cell lines to stimulating peptide was determined by cytotoxic assays using peptide pulsed C1R:A2 or C1R:A3 cells. The CTL were phenotypically CD4+/CD8+ or CD4+/CD8+ and CD56-. The PSA-1, PSA-3 and PSA-OP specific CTLs lysed the HLA-A2 and PSA positive LNCaP prostate carcinoma cells. These studies demonstrate for the first time the ability of human peptides to be capable of generating human CTL lines, and the ability of human prostate carcinoma cells to endogenously process PSA to present specific PSA peptides in the context of MHC class I for T-cell lysis. Recognition by CTLs suggest that PSA peptides may be a potential candidate for use in peptide-based vaccines for prostate cancers.